Plasminogen activators (PA's) are known to play a central role in the fibrinolytic process. Their mechanism of action involves conversion of the inactive proenzyme plasminogen to plasmin, an active serine protease, which in turn degrades the fibrin network of a clot. There are two known types of physiologically relevant PA's: urokinase-type plasminogen activators (uPA) and tissue-type plasminogen activators (TPA). TPA's are found to be far more specific in their action than uPA's showing a higher affinity for fibrin than uPA's and a specificity for the blood clot itself.
Tissue plasminogen activator (TPA) is a glycosylated 66,000 daltons serum protease of 527 amino acid residues produced by the vascular endothelial cells. It is a single polypeptide chain made up of five major domains: the fibronectin finger domain (F.sub.t), the growth factor domain (G.sub.t), the kringle 1 (K.sub.t 1) and kringle 2 (K.sub.t 2) domains and the active site (A.sub.t). It is also known that TPA becomes activated upon binding to fibrin. When digested with plasmin, the one-chain TPA is cleaved at a single site (Arg 275-Ile 276) converting it to a two-chain form, a light and a heavy chain connected by a disulfide bridge. The light chain derived from the carboxy terminus contains the catalytic site. It has a great deal of homology with other serine proteases and does not bind fibrin. The heavy chain derived from the amino terminus is a multi-domain entity with homology to several other serine proteins. It contains a finger domain, a growth factor domain and two kringles. The kringles are triple disulfide structures. The heavy chain has been found to bind fibrin. TPA is cleared by the liver through the heavy chain.
Our recent results have shown that fibrin binding of TPA is localized in two domains. The finger domain contains a high affinity fibrin binding site and the kringle 2 domain a low affinity fibrin binding site. The affinity of the latter site is increased by plasminogen. Stimulation by fibrin and fibrinogen fragments however is mainly localized in the kringle 2 domain. Results obtained by another group (van Zonneveld and Pannekoek, CLB, Amsterdam) suggest stimulation by fibrin does not occur when K1 is directly coupled to the light chain. These results lead to the conclusion that the position of kringle 2 (adjacent to the light chain) and its structure are essential for its role in fibrin stimulation of activity. When we compare the amino acid sequences of TPA K2 with TPA K1, the uPA kringle, and the five plasminogen kringles there are only a limited number of amino acids that are unique for TPA K2. Particularly a group of amino acids around number 250 in the inner loop of the kringle is interesting in this respect.
Urokinase was first isolated from urine. It was then isolated from cultured cells, e.g., kidney cell lines and recently by expression of cDNA in E. coli or mammalian cells in culture. When urokinase is expressed in E. coli, it must be renatured.
Urokinase is a single polypeptide of 411 amino acid residues with a molecular weight of 55,000 daltons. This single chain form, also designated pro-urokinase (pro-u-PA) or single chain urokinase (scu-PA) has low activity, but it can be converted to a two-chain (tuc-PA) form by e.g., plasmin. The two chains stay connected to each other by a single disulfide bridge. In u-PA, three domains can be discerned - a growth factor (G.sub.u), a kringle domain (Ku) and a protease domain (P.sub.u).
The G.sub.u domain can interact with a cellular receptor. The P.sub.u domain contains the active site residues serine, histidine, and asparagine which are usually found in serine proteases. The function of the k.sub.u domain is unknown.
Homologous kringle domains also occur in plasminogen, factor XII, prothrombin and lipoprotein. In both t-PA and plasminogen, one or more kringle domains are involved in fibrin binding. This has not been observed in u-PA. Like t-PA, u-PA catalyses the conversion of the inactive proenzyme plasminogen to the active protease plasminogen. In the case of t-PA this conversion is greatly enhanced by fibrin, whereas for u-PA this is not the case. Nevertheless, fibrin specificity for u-PA has been observed. This observation has been explained by assuming that u-PA preferentially activates fibrin-bound plasminogen.
Since the fibrin specificity of t-PA and u-PA is based on a different mechanism, it should be possible in principle to combine the desired properties of both molecules in a single hybrid molecule. Such hybrids have been constructed, containing long pieces of u-PA and the fibrin binding domains of t-PA or plasminogen. The results are disappointing. Although some fibrin affinity is present in some hybirds, they do not compare to that of t-PA itself.
What is needed are novel t-PA and u-PA analogs which have a greater affinity for fibrin clots and a longer half-life.